Abstract G-protein Coupled Receptor (GPCR) signaling pathways are central to cell-cell communication and cellular detection of the external environment. Conserved signaling pathways are used to mediate such diverse processes as hormone signaling, immune function, and cancer progression in humans and mating in yeast. Negative regulation of these pathways by Regulators of G-protein Signaling (RGS) proteins is critical to maintaining cellular function in the presence of continuous extracellular signal. The yeast RGS, Sst2, is cytosolic during vegetative growth when the pheromone pathway is not active. Upon activation of the GPCR, Ste2, the RGS binds to receptors at the center of polarized growth, but not the edges, and also to septin structures that form at the base of the mating projection. The mechanisms that regulate these interactions are not understood. We hypothesize that dynamic control of RGS subcellular localization serves A) to enhance initial response to pheromone while maintaining strong desensitization of the pathway later in the response, and B) to provide spatial regulation of the pathway to enhance yeast mating. We will use live cell imaging, biochemistry, and yeast genetics approaches to test this hypothesis. In aim 1, we will examine the effect of dynamic RGS association with the receptor and septins and how these interaction impact sensitivity of the pathway over time, morphogenesis, polarization, and mating. In aim 2, we will identify the mechanism by which Sst2 interaction with the receptor and with septins are regulated. Dynamic control of RGS-Receptor interactions is common in humans, and yeast provides an excellent system to uncover the impacts this type of signaling motif has on cell physiology.